While the detailed mechanisms of tumorigenesis are unknown, increasing evidence suggests that genetic alterations of cellular oncogenes are, in part, responsible for the neoplastic transformation of cells. Some studies in rodent models have implicated the direct chemical activation of oncogenes by carcinogen exposure. The reproducible detection of specific transforming genes in animal model systems strongly suggests that these genes have a significant role in the development of certain tumors. Data from our preliminary transfection experiments with DNA from carcinogen-induced medaka tumors support this hypothesis. Our data suggest that an unknown gene may be activated in the DEN-induced cholangiocarcinoma. It does not appear to have homology to any known oncogene sequence based on hybridization of Southern blots. Parallel secondary and tertiary transfections without the addition of pSV2neo revealed the presence of plasmid sequences in some transfected cells, highly correlated with cell transformation. This suggested that pSV2neo may be integrated into the host genome and tightly physically linked to the putative fish oncogene, and may be used as a marker to identify this gene. The identification and characterization of this apparently novel oncogene is now in progress. A library was prepared from EcoRI partial digests of tertiary transfectant DNA ligated into an EMBL 4 vector. The library was screened with pSV2neo; 12 positive clones were isolated and are now being characterized. Studies have also been initiated to develop transgenic medaka by the introduction of the E. coli lacZ gene under the control of the mouse Mc-I metallothionein promoter. Gene expression will be induced by exposure of the fish to heavy metals and analyzed by a colorimetric assay using the indigogenic substrate, X-gal. This construct has also been introduced into fish cell lines to form the basis of in vitro toxicity assays. Preliminary determinations of levels of metals toxic to several cell lines are in progress. These transfectants are now being analyzed.